(418bx) Biomimetic Topographical Replicas of Small Intestine for Regenerative Medicine and Drug Delivery Platforms

Introduction: Diseases and disorders of the intestinal tract affect millions of
people worldwide each year. Inflammatory bowel disease (IBD) represents the
leading chronic gastrointestinal disease with increasing healthcare burdens and
expenditures [1]. There are over 1.4 million persons living with IBD in the
U.S. alone, and as there is no cure, this number increases yearly indicating a
need for new therapeutic strategies [2]. Current in vitro models of IBD utilize
2D systems and Caco-2 epithelial cell lines with a limited ability to mimic
complex tissue. Native small intestine possesses distinct structures (e.g.,
crypts-villi) and a heterogeneous cell population (stem, absorptive, goblet, enteroendocrine, and paneth cells). Therefore, we propose
to develop functional engineered small intestine with relevant heterogeneous
cell populations and spatial organization required for successful regenerative
medicine and drug discovery platforms. Recent breakthroughs have enabled
long-term primary intestinal culture in the form of enteroids, but size
limitations and lack of access to luminal surfaces limits practical
applications [3]. Recent work also suggests cell fate is impacted by 3D
features and local topography/stiffness [4,5]. Therefore, we hypothesize that
incorporating the 3D topography of native intestine into growth substrates with
tunable mechanical and adhesion properties will enable dissociated native
primary enteroid spatial organization similar to native tissue in a scalable
format.

Materials and Methods: To fabricate structurally biomimetic growth
substrates, fresh porcine small intestine was rinsed, fixed with 1% GTA: 1% PFA
in 0.1M PBS (4¬°C, 24hrs), sectioned (1cm^2), placed in 0.1% OsO4 in 0.1M PBS
(4¬°C, 72hrs), and agitated to remove the epithelium. Samples were rinsed,
ethanol dehydrated, critically point dried, treated with silane
(APTS, 12 hrs), and 10:1 v:v PDMS was solution cast for 12hrs followed by
baking (60¬°C, 2hrs). The PDMS negative mold was released from the tissue via
soaking in TEA (12hrs, 25¬°C), and rinsed in 100% ethanol (25¬°C, 24hrs) and
DIH2O (25¬°C, 24hrs), prior to drying (75¬°C, 2hrs). Samples were visualized with
a Hitachi S-4800 UHR field emission SEM [6]. Collagen-I:10%
Matrigel (v:v) hydrogels were cast into 24-well
plates with the PDMS negative replicas or flat surfaces and allowed to gel (2 hrs, 37^oC). Primary mouse enteroids from
isolated crypts [7] were dissociated and seeded on replica hydrogels or flat
controls for 5 days under standard culture conditions. On day 6, samples were
incubated with EdU for 24 hrs. Samples were then
fixed, stained for alpha-tubulin cytoskeleton and nuclei (Hoechst), and imaged
with a Nikon A1R confocal at 20x.

Results and Discussion: Native small intestinal tissue molds were used as
templates to fabricate biomimetic PDMS and hydrogel replicas with irregular,
but spatially organized multi-scale topographical features. Fixed native tissue
and topographical replicas show micro to nano-scale features, containing
irregular, but spatially organized crypt-villus structures and smaller features
of the epithelial basement membrane (not shown). Preliminary qualitative
results indicate that cells positive for EdU
(indicating DNA synthesis) are present in higher amounts on topographical
replicas compared to flat hydrogel controls (Fig. 1). On topographical
replicas, the proliferating cells appear in dense clusters with satellite EdU+ cells throughout the population, whereas on flat
substrates there are sparse EdU+ cells throughout the
monolayer. Further, microtubules appear to localize to the apical surface,
indicating polarity, on replicas but not flat controls. Previous work in our
lab has show that crypt-like structures aid Caco-2 differentiation 8,9,
indicating the importance of topography in epithelial differentiation and
similar effects may be apparent for primary enteroids.

Conclusions:
Manipulating primary enteroid spatial organization and differentiation into
functional models of native small intestine via presentation of biomimetic
topographical cues may provide a better system for regenerative medicine and
drug delivery and discovery applications. This work shows the ability to
engineer growth substrates that mimic the native intestinal multi-scale
topography, and these substrates may impact primary enteroid cell morphology
and proliferation. Future work will determine the impact of physical properties
(e.g., stiffness), chemical properties (e.g., substrate) and structure (e.g.,
intestinal topography) on cell behavior (e.g., attachment, signaling,
differentiation and spatial organization) for a molecular understanding of how
biomimetic topography impacts phenotypic changes.

Acknowledgements: Northeastern STEM Future Faculty Fellowship (ANK), and NIH R21EY021312
(RLK). 

References:
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Cell Mater. 2009;18(1-13):13-14. 5) Reilly GC J Biomech. 2010;43(1):55-62. 6) Pfluger CA, Biomacromol. 2010;11(6):1579–1584. 7) Sato T, Nature. 2009;459(7244):262-265. 8) Wang L, Biomaterials. 2010;31(29):7586-7598. 9) Wang L, Biomaterials. Dec 2009;30(36):6825-6834.